Expression of multiple transgenes within the same target cells is required for several gene transfer and therapy applications1. Gene-function studies are best performed by expressing cDNAs together with a marker gene; by this approach, genetically-modified cells can be identified and monitored in vitro and in vivo. Similarly, gene therapy applications can be improved by purification of gene-corrected cells before in vivo administration, taking advantage of coordinate expression of selectable markers. Genetically-modified cells can be amplified ex vivo or in vivo by introducing growth-promoting or drug-resistance genes together with the therapeutic gene, as recently shown by MGMT-mediated selection of transduced Hematopoietic Stem Cells (HSC)2; using this approach, the efficacy of gene therapy can be increased, and its application potentially extended to a wide spectrum of diseases3, 4. Conversely, genetically-modified cells expressing conditionally cytotoxic genes, together with the therapeutic gene, can be eliminated in vivo, if adverse events occur; this approach is used to control graft-versus-host disease following donor T-lymphocytes infusion to treat leukemia relapse5; it may also provide an important safety provision in HSC gene transfer, given the recent occurrence of leukemia related to vector integration in a successful clinical trial of X-linked Severe Combined ImmunoDeficiency6. Coordinate expression of more than one transgene is essential when the activity to be reconstituted by gene transfer depends on multiple subunits encoded by different genes, or requires the synergism of separate molecules. For instance, reconstitution of the dopamine biosynthetic pathway in striatal neurons of Parkinson's disease patients requires co-expression of tyrosine hydroxylase with GTp-cyclohydrolase I and/or DOPA decarboxylase7; cancer gene therapy may require co-expression of multiple antigens and/or cytokines in antigen-presenting cells for immunotherapy, and of two T-cell receptor chains in T-cells engineered for adoptive transfer8.
In spite of such well-recognized needs, reaching coordinate, high-level expression of multiple transgenes in the majority of target cells has been a significant challenge for gene transfer technology. Two different transgenes have been expressed by two separate vectors; yet, only a fraction of target cells was transduced by both vectors and a heterogeneous population of cells was obtained that expressed either one or two genes in different ratios, preventing reliable studies and/or efficacious applications. Alternatively, two or more transgenes have been expressed by different promoters within the same vector9; yet, different tissue specificity and mutual interference between promoters often prevented efficient co-expression in the same target cells10. Differential splicing generates multiple transcripts from the same promoter, but it has proven difficult to adapt to viral delivery of multiple transgenes11. Chimeric polyproteins that self-process co-translationally into separate components have been generated using the self-cleaving peptide of the Foot and Mouth Disease Virus 2A12, 13; however, application of this technology to multiple gene transfer has been limited until now because it requires sophisticated engineering, restricts both proteins to the same cellular compartment, and introduces sequence changes that may affect protein activity, stability, and immunogenicity. The most satisfactory approach to multiple gene transfer until now has relied on using internal ribosome entry sites (IRES's)14. These sequences, identified in viral and cellular transcripts, control translation in a mRNACap-independent manner and, when inserted between two genes in a bicistronic messenger RNA, allow translation of the downstream gene. The authors tested the performance of different IRES's in the context of self-inactivating (SIN) lentiviral vectors (LVs), and found significant limitations of this approach.
WO 02/064804 describes bi-directional dual promoter complexes that are effective for enhancing transcriptional activity of transgenes in plants.
The bi-directional promoters of the invention include a modified enhancer region with at least two core promoters on either side of the modified enhancer in a divergent orientation. The application refers to gene expression in plants. In addition, the approach requires the duplication of tandem oriented enhancer sequences in a modified internal region of the construct, to be joined by two identical or homologous minimal promoters on either sides. The instant invention does not require duplication of enhancer or any other sequences in the efficient promoter of the bi-directional construct, nor are need that the core promoters on either sides of it to share at least 30% identity. Finally, tandem duplication may be incompatible with retro/lentiviral delivery.
U.S. Pat. No. 6,388,170 discloses plant vectors, having bi-directional promoters, comprising a minimal promoter and a common promoter, wherein said minimal promoters is operably linked to said common promoter, in opposite orientation to said common promoter, and 5′ to said common promoter. Promoter sequences derived from plants and plant-infecting viruses are disclosed dnd tested in plant cells or plant parts. Given the substantial evolutionary distance between plants and animals, U.S. Pat. No. 6,388,170 does not teach how to engineer animal promoters for bi-directional activity and whether bi-directional promoters may effectively work in animal cells. In addition, U.S. Pat. No. 6,388,170 does not teach how to engineer bi-directional promoters for gene expression in animals and in animal cells using the available gene transfer methods.
WO01/34825 discloses cell lines, plasmids and vectors useful for the production of recombinant viruses such as adenoviruses, which are useful in gene therapy. The cell lines, plasmids and vectors comprise inducible promoters, such as bi-directional promoters for the coordinate expression of bidirectionally cloned gene. However only bi-directional Tet-regulated constructs are disclosed.
Thus, the authors explored novel strategies to take full advantage of gene transfer systems, such as LV, that allow efficient ex vivo transduction and direct in vivo administration.